Mucosal surfaces are the main site of entry of pathogens, and some of the most dangerous pathogens enter their host and initiate the infection at such sites. Thus, the upper-respiratory, gastro-intestinal or urogenital-tracts remain the prime target of pathogens. Targeting an infection at the initiation site, which would contain and prevent mucosal invasion of the pathogen, neutralize the pathogen-derived toxin and inhibit the replication of the pathogen within the body at later infections stages, is undeniably the most important feature of mucosal vaccines (Holmgren, J., and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nat. Med 11:S45-S53). Therefore, mucosal immunization and the development of mucosal vaccines remains the Holy Grail for all vaccinologists. Furthermore, there are additional advantages of using mucosal vaccines which are multiple and obvious. Cheaper costs, needle-free delivery, safety, ease of administration, especially in case of mass immunization during pandemics, or lack of trained personnel, make mucosal vaccines very attractive compared to the traditional injectable vaccines. In addition, mucosal vaccination has the capacity to induce protective immunity in both the systemic and mucosal compartments, which classical parenteral vaccination fails to do, therefore providing dual protection (Fujkuyama, Y., D. Tokuhara, K. Kataoka, R. S. Gilbert, J. R. McGhee, Y. Yuki, H. Kiyono, and K. Fujihashi. 2012. Novel vaccine development strategies for inducing mucosal immunity. Expert. Rev. Vaccines. 11:367). It is also well established that the entire mucosal immune system is immunologically connected, and there is a notion of a “common mucosal immune system” (Brandtzaeg, P. 2007. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 25:5467). Hence it is possible to immunize at one mucosal site whilst inducing immunity at another very distant mucosal site. The best example illustrating this approach remains vaccination via the intra-nasal (i.n.) route, which can elicit protective immunity in the urogenital tract in models of HSV (Gallichan, W. S., and K. L. Rosenthal. 1998. Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis 177:1155), and HIV infections (Gherardi, M. M., E. Perez-Jimenez, J. L. Najera, and M. Esteban. 2004. Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus Ankara boost immunization schedule. J Immunol. 172:6209).
Nevertheless, despite all these advantages of mucosal vaccines, very few have made it to the market, compared to the parenteral vaccines. Those that have made it to the market for humans, are based on live-attenuated or heat-killed whole-cell vaccines, which include the oral cholera vaccine, the oral polio vaccine, the oral typhoid vaccine, the rotavirus vaccine for infants, the nasal-spray influenza vaccine. Due to some complications and adverse side-effects, some of them had to be withdrawn from the market, such as the seasonal 2001-H1N1 influenza vaccine, which induced facial paralysis in some patients in Switzerland (Mutsch, M., W. Zhou, P. Rhodes, M. Bopp, R. T. Chen, T. Linder, C. Spyr, and R. Steffen. 2004. Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland. N. Engl. J Med 350:896). Therefore, there are also concerns and safety measures, which need to be taken into consideration when developing a mucosal vaccine. Another important aspect of mucosal vaccines, is that they remain less immunogenic, compared to their traditional counterparts. Therefore the need for adjuvants is given, if one wants to break the tolerogenic environment, in the case of oral immunization, and induce more potent protective and long-lasting immune responses. In recent years, new research advances have been made in the mucosal immunology field and there is now a better understanding of the immunological mechanisms occurring at the mucosal surfaces. Even though there is still a long way to go, this intensive research has been a great step on the way for finding the best mucosal adjuvants and delivery systems, tailored for each pathogen.
The use of adjuvant is essential in vaccine research, especially when using recombinant sub-unit vaccines which remain less immunogenic compared to their heat-killed or live-attenuated counterparts. Indeed, it is believed that live-attenuated or inactivated pathogen-based vaccines inherently contain adjuvants. For the last 80 years, the only licensed adjuvant used in humans has been Alum (Aluminium salts) and oil-in-water emulsions. Recently two other adjuvants were licensed and can now be used, namely AS04 from GSK (a monophosphory lipid A preparation with aluminium salts) and MF59 (an oil-in-water preparation) used in combination with the seasonal influenza vaccine from Novartis. The disadvantage of oil-in-water emulsions and aluminium salt is that they do not elicit strong mucosal T helper cell responses. Studies have shown that the most promising mucosal adjuvants are bacterial toxin derivatives, Toll-like receptor (TLR) ligands, and novel small molecules (Rhee, J. H., S. E. Lee, and S. Y. Kim. 2012. Mucosal vaccine adjuvants update. Clin Exp. Vaccine Res. 1:50). The function of adjuvants is to boost the innate arm of the immune system, as they act mainly on antigen-presenting cells (APCs), such as dendritic cells (DCs), which in turn improve T and B cell responses to antigens (Schijns, V. E. 2001. Induction and direction of immune responses by vaccine adjuvants. Crit Rev. Immunol. 21:75). Pathogen associated molecular patterns (PAMPs), recognised by pattern recognition receptors (PRRs), are very potent adjuvants which strongly stimulate innate immune cells. PRRs are expressed on the cell surface or within intracellular compartments. Members of the PRR family include TLRs, nucleotide-binding domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene (RIG)-like receptors (RLRs), and C-type lectins (Akira, S. 2011. Innate immunity and adjuvants. Philos. Trans R. Soc Lond B Biol. Sci 366:2748). Therefore, the use of TLR-based adjuvants, including bacterial cell wall lipopeptides (TLR4, TLR2) (Duthie, M. S., H. P. Windish, C. B. Fox, and S. G. Reed. 2011. Use of defined TLR ligands as adjuvants within human vaccines. Immunol. Rev. 239:178), and CpG motifs of bacterial DNA (TLR9) (Bode, C., G. Zhao, F. Steinhagen, T. Kinjo, and D. M. Klinman. 2011. CpG DNA as a vaccine adjuvant. Expert. Rev. Vaccines. 10:499) have been the adjuvants of choice to be incorporated in the new generation vaccines.
As mentioned previously, adjuvants are becoming an essential part for the development of mucosal vaccines. The TLR5 ligand flagellin, the major structural protein of Gram-negative flagella involved in the motility of bacteria, has elicited a lot of interest, and studies going back nearly a decade have been described using flagellin as a potent adjuvant, in the context of a broad range of recombinant vaccines (Mizel, S. B., and J. Bates. 2014. Flagellin as an adjuvant: cellular mechanisms and potential, pp. 5677). Flagellin has been used separately with the antigen or commonly administered as fusion proteins, which have proven to be very effective vaccines in animal models of influenza (Wang, B. Z., R. Xu, F. S. Quan, S. M. Kang, L. Wang, and R. W. Compans. 2010. Intranasal immunization with influenza VLPs incorporating membrane-anchored flagellin induces strong heterosubtypic protection. PLoS. One. 5:e13972), Yersinia Pestis (Honko, A. N., N. Sriranganathan, C. J. Lees, and S. B. Mizel. 2006. Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis. Infect Immun. 74:1113), West nile virus (McDonald, W. F., J. W. Huleatt, H. G. Foellmer, D. Hewitt, J. Tang, P. Desai, A. Price, A. Jacobs, V. N. Takahashi, Y. Huang, V. Nakaar, L. Alexopoulou, E. Fikrig, and T. J. Powell. 2007. A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity. J Infect Dis 195:1607), Pseudomonas aeruginosa (Weimer, E. T., H. Lu, N. D. Kock, D. J. Wozniak, and S. B. Mizel. 2009. A fusion protein vaccine containing OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid Pseudomonas aeruginosa. Infect Immun. 77:2356), Plasmodium falciparum (Carapau, D., R. Mitchell, A. Nacer, A. Shaw, C. Othoro, U. Frevert, and E. Nardin. 2013. Protective humoral immunity elicited by a needle-free malaria vaccine comprised of a chimeric Plasmodium falciparum circumsporozoite protein and a Toll-like receptor 5 agonist, flagellin. Infect Immun. 81:4350) and Vaccinia virus (Delaney, K. N., J. P. Phipps, J. B. Johnson, and S. B. Mizel. 2010. A recombinant flagellin-poxvirus fusion protein vaccine elicits complement-dependent protection against respiratory challenge with vaccinia virus in mice. Viral Immunol. 23:201). The effect of flagellin occurs on various APCs, DCs, neutrophils, monocytes, and more specifically on airway structural epithelial cells (Van, M. L., D. Fougeron, L. Janot, A. Didierlaurent, D. Cayet, J. Tabareau, M. Rumbo, S. Corvo-Chamaillard, S. Boulenouar, S. Jeffs, W. L. Vande, M. Lamkanfi, Y. Lemoine, F. Erard, D. Hot, T. Hussell, B. Ryffel, A. G. Benecke, and J. C. Sirard. 2014. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal. Immunol. 7:489) and enterocytes (Van, M. L., D. Fougeron, L. Janot, A. Didierlaurent, D. Cayet, J. Tabareau, M. Rumbo, S. Corvo-Chamaillard, S. Boulenouar, S. Jeffs, W. L. Vande, M. Lamkanfi, Y. Lemoine, F. Erard, D. Hot, T. Hussell, B. Ryffel, A. G. Benecke, and J. C. Sirard. 2014. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal. Immunol. 7:489 and Gewirtz, A. T., T. A. Navas, S. Lyons, P. J. Godowski, and J. L. Madara. 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol. 167:1882). The latter have been shown to produce chemokines following TLR recognition, which in turn recruit more APCs to the site of immunization. The binding of flagellin to TLR5 initiates an immune cascade leading to production of the pro-inflammatory cytokines, IL-6 and TNF-α (Raoust, E., V. Balloy, I. Garcia-Verdugo, L. Touqui, R. Ramphal, and M. Chignard. 2009. Pseudomonas aeruginosa LPS or flagellin are sufficient to activate TLR-dependent signaling in murine alveolar macrophages and airway epithelial cells. PLoS. One. 4:e7259). In addition, another recognition pathway used by flagellin involves the Nlrc-4-inflammosome pathway (Miao, E. A., and S. E. Warren. 2010. Innate immune detection of bacterial virulence factors via the NLRC4 inflammasome. J Clin Immunol. 30:502), a member of the intracellular (NLR) family, which in this case leads to the production of IL-1β and IL-18 (Kupz, A., G. Guarda, T. Gebhardt, L. E. Sander, K. R. Short, D. A. Diavatopoulos, O. L. Wijburg, H. Cao, J. C. Waithman, W. Chen, D. Fernandez-Ruiz, P. G. Whitney, W. R. Heath, R. Curtiss, III, J. Tschopp, R. A. Strugnell, and S. Bedoui. 2012. NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8(+) T cells. Nat. Immunol. 13:162). In terms of adaptive immunity, it is well recognized that flagellin induces the proliferation of antigen-specific CD4+ T cells (Bates, J. T., S. Uematsu, S. Akira, and S. B. Mizel. 2009. Direct stimulation of tlr5+/+ CD11c+ cells is necessary for the adjuvant activity of flagellin. J Immunol. 182:7539), together with robust antibody responses, characterized with high IgG1 and IgG2 titers (Huleatt, J. W., A. R. Jacobs, J. Tang, P. Desai, E. B. Kopp, Y. Huang, L. Song, V. Nakaar, and T. J. Powell. 2007. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25:763). It is believed that the adjuvant effect of flagellin on adaptive immunity is enhanced when administered as flagellin-antigen fusion proteins.
Among the many possible vaccine modalities, live vector viruses seem best to fulfill the requirement for inducing both T-cell and B-cell responses (R. A. Koup and D. C. Douek, “Vaccine Design for CD8+ T Lymphocyte Responses,” Cold Spring Harb. Perspect. Med. 2011; 1:a007252). While some live virus vaccines, such as vaccinia virus and yellow fever virus are effective but have unfavorable safety profiles, others, such as adenovirus, face problems due to pre-existing immunity (A. R. Thorner, et al., “Age Dependence of Adenovirus-Specific Neutralizing Antibody Titers in Individuals from Sub-Saharan Africa,” J. Clin. Microbiol. 44(10):3781-3783 (2006)). A safe live vector vaccine unaffected by preexisting immunity is modified vaccinia virus Ankara (MVA), originally created by Anton Mayr and further developed into a third-generation smallpox vaccine (MVA-BN®) (Kennedy J S, Greenberg R N IMVAMUNE: modified vaccinia Ankara strain as an attenuated smallpox vaccine. Expert Rev Vaccines. 2009 January; 8(1):13-24. doi: 10.1586/14760584.8.1.13. Review).
The excellent safety profile of MVA, because of its replication deficiency in human cells, has been proven in many clinical trials, including vaccination of immune-compromised individuals, and during the smallpox eradication campaign in the 1970s, when 120,000 people were vaccinated with MVA (A. Mayr et al., “The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defense mechanism,” Zentralbl. Bakteriol. B 167(5-6):375-390 (1978)). Since then, many different recombinant MVA vaccines have been generated and tested for the ability to immunize animals and humans against infectious (e.g., HIV, malaria) and non-infectious (e.g., prostate cancer) diseases. Its proven safety and good immunogenicity thus make MVA a prime candidate for a T- and B-cell-inducing vaccine vector.
Most studies using recombinant MVA have been described after systemic application. Nevertheless, a few reports have examined the immune response after mucosal administration of MVA, especially i.n. delivery in studies of Influenza (Sutter, G., L. S. Wyatt, P. L. Foley, J. R. Bennink, and B. Moss. 1994. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12:1032.), RSV (Wyatt, L. S., S. T. Shors, B. R. Murphy, and B. Moss. 1996. Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine 14:1451) and HIV (Gherardi, M. M., E. Perez-Jimenez, J. L. Najera, and M. Esteban. 2004. Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus Ankara boost immunization schedule. J Immunol. 172:6209). One study describing the role of a flagellin-poxvirus vaccine, in this case using recombinant vaccinia virus antigen proteins, L1R and B5R, fused to flagellin, demonstrated a protective effect in mice immunized with this flagellin-fusion protein against a vaccinia virus challenge (Delaney, K. N., J. P. Phipps, J. B. Johnson, and S. B. Mizel. 2010. A recombinant flagellin-poxvirus fusion protein vaccine elicits complement-dependent protection against respiratory challenge with vaccinia virus in mice. Viral Immunol. 23:201). Furthermore, the use of flagellin delivered via the i.n. route, has been used in a number of studies in various mouse models and in non-human primates at a very low dose (Weimer, E. T., S. E. Ervin, D. J. Wozniak, and S. B. Mizel. 2009. Immunization of young African green monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the production of protective antibodies against nonmucoid Pseudomonas aeruginosa. Vaccine 27:6762), making it a very attractive mucosal adjuvant candidate that could be used in human vaccines. To date, there are no reports using rMVA together with flagellin, particularly genetically encoded within the vector, and administered via the i.n. route